The unfavorable clinical outcome of COVID-19 in smokers is mediated by H3K4me3, H3K9me3 and H3K27me3 histone marks

Smoking could predispose individuals to a more severe COVID-19 by upregulating a particular gene known as mdig, which is mediated through a number of well-known histone modifications. Smoking might regulate the transcription-activating H3K4me3 mark, along with the transcription-repressing H3K9me3 and H3K27me3 marks, in a way to favor SARS-CoV-2 entry by enhancing the expression of ACE2, NRP1 and NRP2, AT1R, CTSD and CTSL, PGE2 receptors 2–4, SLC6A20 and IL-6, all of which interact either directly or indirectly with important receptors, facilitating viral entry in COVID-19.

Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic "hit". Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability. Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression. Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1. Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

HMGA1 Chromatin Regulators Drive Progression in Myeloproliferative Neoplasms through Epigenetic Rewiring to Induce Networks Involved in GATA2 and Proliferation

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemic transformation. The High Mobility Group A1 (HMGA1) gene encodes oncogenic chromatin regulators which are overexpressed in diverse tumors where they portend adverse outcomes (Resar Cancer Res 2010; Xian et al Nature Commun 2017). Hmga1 induces leukemic transformation in transgenic mice and HMGA1 is overexpressed in refractory myeloid malignancies (Resar et al Cancer Res 2018). Further, germline lesions within the HMGA1 loci increase the risk for developing MPN (Bao et al Nature 2020). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by rewiring transcriptional networks to foster leukemogenesis, and, 2) identify mechanisms underlying HMGA1 that could be targeted with therapy. Methods: To elucidate the function of HMGA1, we disrupted HMGA1 expression via CRISPR/Cas9 or short hairpin RNA (shRNA) targeting 2 different sequences per gene and assessed proliferation, colony formation, apoptosis, and leukemogenesis. We also generated JAK2 V617F transgenic mouse models of MF with Hmga1 deficiency. To dissect molecular mechanisms underlying HMGA1, we integrated RNAseq, ATACseq, and chromatin immunoprecipitation (ChIP) from MPN-AML cell lines (DAMI, SET-2). Next, we tested whether HMGA1 depletion synergizes with ruxolitinib in preventing leukemic engraftment in mice. To identify drugs to target HMGA1 networks, we applied the Broad Institute Connectivity Map (CMAP). Results: HMGA1 is overexpressed in CD34 + cells from patients with JAK2 V617F MPN with highest levels after transformation to MF or AML in 3 independent cohorts. CRISPR/Cas9 inactivation or shRNA-mediated HMGA1 silencing disrupts proliferation, decreases the frequency of cells in S phase, increases apoptosis, and impairs clonogenicity in human MPN-AML cell lines. HMGA1 depletion also prevents leukemic engraftment in mice. Surprisingly, loss of just a single Hmga1 allele prevents progression to MF in JAK2 V617Fmurine models of MPN, decreasing erythrocytosis, thrombocytosis, and preventing splenomegaly and fibrosis of the spleen and bone marrow. Further, Hmga1 deficiency preferentially prevents expansion in long-term HSC, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors in JAK2 V617F mice. RNAseq revealed genes induced by HMGA1 that govern cell cycle progression (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) and cell fate decisions (GATA2 networks), including the GATA2 master regulator gene. Silencing GATA2 recapitulates anti-leukemia phenotypes observed with HMGA1 deficiency whereas restoring GATA2 in MPN-AML cells with HMGA1 silencing partially rescues leukemia phenotypes, increasing clonogenicity and leukemic engraftment. Mechanistically, HMGA1 binds directly to AT-rich sequences near the GATA2 developmental enhancer (+9.5), enhances chromatin accessibility, and recruits active histone marks (H3K4me1/3) to induce GATA2 expression. HMGA1 depletion enhances responses to the JAK/STAT Inhibitor, ruxolitinib, delaying leukemic engraftment and prolonging survival in murine models of JAK2 V617F MPN-AML. Further, epigenetic drugs predicted to target HMGA1 transcriptional networks using CMAP synergize with JAK inhibitors to disrupt proliferation in human MPN-AML cells. HMGA1 and GATA2 are co-expressed and up-regulated with progression from MF to AML in matched patient samples. Moreover, HMGA1 transcriptional networks are activated in leukemic blasts, thus underscoring the role of HMGA1 in human MPN progression. Conclusions: We uncovered a previously unknown epigenetic program whereby HMGA1 enhances chromatin accessibility and recruits activating histone marks to induce transcriptional networks required for progression in MPN, including direct transactivation of GATA2. Further, HMGA1 networks can be targeted with epigenetic therapy and synergize with ruxolitinib. Together, our studies reveal a new paradigm whereby HMGA1 up-regulates GATA2 and proliferation networks to drive disease progression and illuminate HMGA1 as a novel therapeutic target in MPN. Figure 1 Figure 1. Disclosures Rampal: Jazz Pharmaceuticals: Consultancy; Incyte: Consultancy, Research Funding; Kartos: Consultancy; Constellation: Research Funding; Pharmaessentia: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; Stemline: Consultancy, Research Funding; BMS/Celgene: Consultancy; Novartis: Consultancy; Sierra Oncology: Consultancy; CTI: Consultancy; Abbvie: Consultancy; Memorial Sloan Kettering: Current Employment. Stubbs: Incyte Research Institute: Current Employment, Current holder of individual stocks in a privately-held company.

Is There a Histone Code for Cellular Quiescence?

Many of the cells in our bodies are quiescent, that is, temporarily not dividing. Under certain physiological conditions such as during tissue repair and maintenance, quiescent cells receive the appropriate stimulus and are induced to enter the cell cycle. The ability of cells to successfully transition into and out of a quiescent state is crucial for many biological processes including wound healing, stem cell maintenance, and immunological responses. Across species and tissues, transcriptional, epigenetic, and chromosomal changes associated with the transition between proliferation and quiescence have been analyzed, and some consistent changes associated with quiescence have been identified. Histone modifications have been shown to play a role in chromatin packing and accessibility, nucleosome mobility, gene expression, and chromosome arrangement. In this review, we critically evaluate the role of different histone marks in these processes during quiescence entry and exit. We consider different model systems for quiescence, each of the most frequently monitored candidate histone marks, and the role of their writers, erasers and readers. We highlight data that support these marks contributing to the changes observed with quiescence. We specifically ask whether there is a quiescence histone “code,” a mechanism whereby the language encoded by specific combinations of histone marks is read and relayed downstream to modulate cell state and function. We conclude by highlighting emerging technologies that can be applied to gain greater insight into the role of a histone code for quiescence.

The Paramount Role of Drosophila melanogaster in the Study of Epigenetics: From Simple Phenotypes to Molecular Dissection and Higher-Order Genome Organization

Drosophila melanogaster has played a paramount role in epigenetics, the study of changes in gene function inherited through mitosis or meiosis that are not due to changes in the DNA sequence. By analyzing simple phenotypes, such as the bristle position or cuticle pigmentation, as read-outs of regulatory processes, the identification of mutated genes led to the discovery of major chromatin regulators. These are often conserved in distantly related organisms such as vertebrates or even plants. Many of them deposit, recognize, or erase post-translational modifications on histones (histone marks). Others are members of chromatin remodeling complexes that move, eject, or exchange nucleosomes. We review the role of D. melanogaster research in three epigenetic fields: Heterochromatin formation and maintenance, the repression of transposable elements by piRNAs, and the regulation of gene expression by the antagonistic Polycomb and Trithorax complexes. We then describe how genetic tools available in D. melanogaster allowed to examine the role of histone marks and show that some histone marks are dispensable for gene regulation, whereas others play essential roles. Next, we describe how D. melanogaster has been particularly important in defining chromatin types, higher-order chromatin structures, and their dynamic changes during development. Lastly, we discuss the role of epigenetics in a changing environment.

The Spin1 interactor, Spindoc, is dispensable for meiotic division, but essential for haploid spermatid development in mice

In mammals, germline development undergoes dramatic morphological and molecular changes and is epigenetically subject to intricate yet exquisite regulation. Which epigenetic players and how they participate in the germline developmental process are not fully characterized. Spin1 is a multifunctional epigenetic protein reader that has been shown to recognize H3 “K4me3-R8me2a” histone marks, and more recently the non-canonical bivalent H3 “K4me3-K9me3/2” marks as well. As a robust Spin1-interacting cofactor, Spindoc has been identified to enhance the binding of Spin1 to its substrate histone marks, thereby modulating the downstream signaling; However, the physiological role of Spindoc in germline development is unknown. We generated two Spindoc knockout mouse models through CRISPR/Cas9 strategy, which revealed that Spindoc is specifically required for haploid spermatid development, but not essential for meiotic divisions in spermatocytes. This study unveiled a new epigenetic player that participates in haploid germline development.

Region-specific H3K9me3 gain in aged somatic tissues in Caenorhabditis elegans

Epigenetic alterations occur as organisms age, and lead to chromatin deterioration, loss of transcriptional silencing and genomic instability. Dysregulation of the epigenome has been associated with increased susceptibility to age-related disorders. In this study, we aimed to characterize the age-dependent changes of the epigenome and, in turn, to understand epigenetic processes that drive aging phenotypes. We focused on the aging-associated changes in the repressive histone marks H3K9me3 and H3K27me3 in C. elegans. We observed region-specific gain and loss of both histone marks, but the changes are more evident for H3K9me3. We further found alteration of heterochromatic boundaries in aged somatic tissues. Interestingly, we discovered that the most statistically significant changes reflected H3K9me3-marked regions that are formed during aging, and are absent in developing worms, which we termed “aging-specific repressive regions” (ASRRs). These ASRRs preferentially occur in genic regions that are marked by high levels of H3K9me2 and H3K36me2 in larval stages. Maintenance of high H3K9me2 levels in these regions have been shown to correlate with a longer lifespan. Next, we examined whether the changes in repressive histone marks lead to de-silencing of repetitive DNA elements, as reported for several other organisms. We observed increased expression of active repetitive DNA elements but not global re-activation of silent repeats in old worms, likely due to the distributed nature of repetitive elements in the C. elegans genome. Intriguingly, CELE45, a putative short interspersed nuclear element (SINE), was greatly overexpressed at old age and upon heat stress. SINEs have been suggested to regulate transcription in response to various cellular stresses in mammals. It is likely that CELE45 RNAs also play roles in stress response and aging in C. elegans. Taken together, our study revealed significant and specific age-dependent changes in repressive histone modifications and repetitive elements, providing important insights into aging biology.

Diet-induced- and genetic- obesity differentially alters male germline histones

Obesity, an established risk factor for male subfertility or infertility, is primarily due to genetic and environmental causes. Our earlier studies have shown differential effects of high fat diet-induced- (DIO) and genetically inherited- (GIO) obesity on DNA methylation in male germline and its subsequent effect on fertility. Here, we hypothesized that the effects of DIO and GIO on histone modifications in male germline could also contribute to fertility defects. We observed that DIO affected both active (H3K4me3, H3ac, and H4ac) and repressive (H3K9me3 and H3K27me3) histone marks in testis and their cell types, whereas GIO solely altered acetylated histones. This correlated with deregulation of histone-modifying enzymes in testis of both obese groups. Further, we also observed a decrease in chromatin remodelers in testis of DIO group, which were increased in GIO group. Besides, there was an increase in core histones and a decrease in histone marks along with protamine deficiency in spermatozoa of DIO group, whereas only H3K4me3 levels were increased in spermatozoa of GIO group. Moreover, we observed alterations in the expression and enrichment patterns of a few developmental genes harbored by the active histone mark in resorbed embryos sired by the DIO rats. Together these epigenetic defects in male germline could alter sperm quality and cause fertility defects in these obese groups. Differential changes in two obese groups could also be attributed to differences in their pathophysiological variations. Our study highlights epigenetic differences between DIO and GIO in male germline and its subsequent impact on male fertility.